Mechanisms of assembly of photoreceptor G protein complexes

 描述(申请人提供)：正确的蛋白质折叠是维持健康神经元的关键问题，包括视网膜的感光细胞。对光感受器蛋白质组的压力可能是由环境因素如光损伤或遗传因素引起的，如视紫红质和许多其他光感受器蛋白质的错误折叠突变。蛋白质组由一类称为分子伴侣的蛋白质维持。这些伴侣保护蛋白质不被聚集，引导它们的折叠途径，并促进它们结合成多蛋白质集合。分子伴侣的一种重要类型是在真核细胞胞浆中发现的II型伴侣蛋白，称为CCT(Cytosolal Chapertin Containing Tailless Complex Polyposal 1，又称TIC)。已知的需要CCT进行折叠的蛋白质数量有数百种，而且还在继续增长。其中包括G蛋白β亚单位(Gβ)，它们构成了Gβγ和Gβ5-RGS(G蛋白信号调节)二聚体，它们是视觉信号中的关键成分。Gβγ和RGS-Gβ5都需要cct辅助伴侣蛋白，即类似光导蛋白的蛋白，才能正确折叠和形成二聚体。在以前的工作中，我们已经通过冷冻电子显微镜、化学交联与质谱联用以及与非天然氨基酸的定点交联等方法确定了Gβγ组装中两个中间体的结构。这些结构为Gβγ组装的机制提供了分子细节。在目标1中，我们建议确定RGS-Gβ5组装中类似中间体的结构，以在分子水平上了解CCT和PhLP1如何协助RGS-Gβ5二聚体的形成。CCT在Bardet-Biedl综合征(BBS)中的另一个作用也已被证明。BBS是一种以视网膜变性等多种病理状态为特征的遗传性睫状体功能障碍疾病。BBS是由于不能形成BBSome引起的，BBSome是一种由八种蛋白质组成的复合体，对于囊泡向纤毛的运输是必不可少的。BBSome的组装需要CCT和由三种已知突变导致BBS的蛋白质(BBS 6、10和12)组成的CCT样复合体。在目标2中，我们提出了类似的结构研究来确定BBSome组装和BBSome功能的分子机制。从这些研究中获得的信息将对设计基于伴侣的方法治疗由RGS-G、β5和BBSome故障引起的视网膜疾病至关重要。

 DESCRIPTION (provided by applicant): Proper protein folding is a key issue in maintaining healthy neurons, including the photoreceptor cells of the retina. Stresses on the photoreceptor proteome may be caused by environmental factors such as light damage or by genetic factors such as misfolding mutations in rhodopsin and many other photoreceptor proteins. The proteome is maintained by a class of proteins called molecular chaperones. These chaperones protect proteins from aggregation, channel their folding pathways and facilitate their association into multi-protein assemblies. An important type of molecular chaperone is the type II chaperonin found in the eukaryotic cytosol, termed CCT (Cytosolic Chaperonin containing Tailless complex polypeptide 1, also called TRiC). The number of proteins known to require CCT for their folding is in the hundreds and continues to grow. Among these are the G protein β subunits (Gβ) which form the Gβγ and Gβ5-RGS (Regulator of G protein Signaling) dimers that are key components in visual signaling. Both Gβγ and RGS-Gβ5 also require the CCT co-chaperone, phosducin-like protein, for proper folding and dimer formation. In previous work, we have determined the structures of two intermediates in Gβγ assembly by cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and site-specific cross-linking with unnatural amino acids. These structures have provided molecular detail into the mechanism of Gβγ assembly. In Aim 1, we propose to determine the structures of similar intermediates in RGS-Gβ5 assembly to understand at the molecular level how CCT and PhLP1 assist in RGS-Gβ5 dimer formation. An additional role for CCT in Bardet-Biedl syndrome (BBS) has also been demonstrated. BBS is a genetic disease of ciliary dysfunction characterized by multiple pathological conditions including retinal degeneration. BBS is caused by an inability to form the BBSome, a complex of eight proteins that is essential for vesicle trafficking to cilia. CCT and a CCT-like complex made up of three proteins whose mutations are known to cause BBS (BBS 6, 10 and 12) are required for the assembly of the BBSome. In Aim 2, we propose similar structural studies to determine the molecular mechanism of BBSome assembly and BBSome function. The information gained from these studies will be vital in designing chaperone-based methods to treat retinal diseases caused by RGS-Gβ5 and BBSome malfunctions.